Methods and compositions for natural killer cells

ABSTRACT

The application provides new compositions and methods for stimulating the production of natural killer (NK) cells in a subject. NK cells can be selectively expanded with a combination of stimulating ligands. Methods and compositions for the administration of stimulatory ligands modified to self-insert into tumor cells, thereby stimulating an increase in the number of NK cells in proximity to a tumor, are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claim priority to U.S.application Ser. No. 15/489,460, filed on Apr. 17, 2017, which is acontinuation of and claims priority to U.S. application Ser. No.14/410,787, filed Dec. 23, 2014, now U.S. Pat. No. 9,623,082, which is a35 U.S.C. § 371 national stage application of PCT Application No.PCT/US2013/048678 filed on Jun. 28, 2013, which claims benefit of U.S.Provisional Application No. 61/665,591, filed Jun. 28, 2012, thedisclosures of which are hereby incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present application generally relates to compositions and methodscomprising natural killer (NK) cells. More particularly, the applicationrelates to the n vivo or in vitro stimulation and expansion ofendogenous natural killer (NK) cells, which are capable of attacking andkilling immune cells and cancer cells.

BACKGROUND

Hematopoietic stem cell transplantation (HSCT) from genotypicallyHLA-matched siblings has improved long-term survival in patients withhematologic cancer malignancies and marrow failure syndromes. Everyyear, more than 10,000 Americans get life-threatening diseases for whichthe only hope of a cure is a bone marrow transplant from an unrelateddonor or cord blood unit. However, more than 70% of patients who couldbenefit from an allogeneic stem cell transplant do not have a matchedsibling donor, (Henslee-Downey, et al. 1997). These circumstances delaytreatment, making it necessary to resort to less than optimal use of apartially mismatched donor, which eventually leads to increasedincidence of graft-versus-host disease (GVHD), graft failure, andrelapse, all of which dramatically decrease patient survival (Drobyski,et al. 2002), (Baker, et al. 2009).

Additional limitations are posed by the duration and the costlyfinancial, mental, and health burdens of the transplant process. Thus,the application of HSCT from an unrelated donor is limited to younger,healthier patients with appropriate socioeconomic support that canendure the process.

Further challenges are posed by the high rate of relapse due to theinability to eradicate residual cancer cells. Although HSCT isconsidered to be curative, cancer relapse rates are staggering. Thus,novel, more targeted immunotherapies are needed that would be moreeffective, preferably without the need for a matched donor. Donorlymphocyte infusion (DLI), for the treatment of acute myeloid leukemia(AML) relapse after HSCT was introduced in 1990s. This approachconsisted of the administration of lymphocytes from the original donorto the AML patient with relapsed disease. Yet, clinical benefits werelimited and observed only in a minority of patients with smaller tumorburdens, and T cell mediated GVHD often further worsened the outcomes.

There is a great need for new and improved methodologies aimed atincreasing NK cell numbers.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF SUMMARY

Disclosed are compositions comprising a plasma membrane vesicle, whereinthe plasma membrane vesicle comprises an NK cell effector agent. NK celleffector agents can be a cytokine, an adhesion molecule, or an NK cellactivating agent.

Disclosed are compositions comprising a plasma membrane vesicle, whereinthe plasma membrane vesicle comprises an NK cell effector agent, whereinthe NK cell effector agent can be IL-15, IL-21, IL-2, 41BBL, IL-12,IL-18, MICA, 2B4, LFA-1, or BCM1/SLAMF2.

Disclosed are compositions comprising a plasma membrane vesicle, whereinthe plasma membrane vesicle comprises an NK cell effector agent, whereinthe NK cell effector agent can be IL-15, IL-21, or 41BBL.

Disclosed are compositions comprising a plasma membrane vesicle, whereinthe plasma membrane vesicle comprises an NK cell effector agent, whereinthe plasma membrane can comprise two or more NK cell effector agents. Atleast one NK cell effector agent can be a cytokine. For example,disclosed are compositions comprising a plasma membrane vesicle, whereinthe plasma membrane vesicle comprises an NK cell effector agent, whereinthe plasma membrane can comprise IL-15 and 41BBL. In some aspects, theplasma membrane can further comprise IL-21. Disclosed are compositionscomprising a plasma membrane vesicle, wherein the plasma membranevesicle comprises an NK cell effector agent, wherein the plasma membranecan comprise IL-21 and 41BBL.

Disclosed are compositions comprising a plasma membrane vesicle, whereinthe plasma membrane vesicle comprises an NK cell effector agent, whereinthe plasma membrane vesicle surrounds a microparticle.

Disclosed are compositions comprising two or more plasma membranevesicles, wherein the plasma membrane vesicles comprise an NK celleffector agent.

Disclosed are compositions comprising at least one compositioncomprising a plasma membrane vesicle comprising an NK cell effectoragent and at least one composition comprising a membrane self-insertingpeptide conjugated to an NK cell effector agent.

Disclosed are compositions comprising a membrane self-inserting peptideconjugated to an NK cell effector agent. NK cell effector agents can bea cytokine, an adhesion molecule, or an NK cell activating agent.Disclosed are compositions comprising a membrane self-inserting peptideconjugated to an NK cell effector agent, wherein the NK cell effectoragent can be IL-15, IL-21, IL-2, 41BBL, IL-12, IL-18, MICA, 2B4, LFA-1,and BCM1/SLAMF2.

Disclosed are compositions comprising a membrane self-inserting peptideconjugated to an NK cell effector agent, wherein the NK cell effectoragent is IL-15, IL-21, or 41BBL.

Disclosed are compositions comprising a membrane self-inserting peptideconjugated to an NK cell effector agent, wherein the membraneself-inserting peptide is a molecule that promotes insertion into amembrane and can be human Fc, GPI, transmembrane T cell receptor, orpHLIP.

Disclosed are compositions comprising two or more compositionscomprising a membrane self-inserting peptide conjugated to an NK celleffector agent.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agent. Atleast one NK cell effector agent can be a cytokine, an adhesionmolecule, or an NK cell activating agent. In some aspects, the NK celleffector agent can be IL-15, IL-21, or 41BBL.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising two or more NK cell effector agents. Atleast one NK cell effector agent can be a cytokine. Thus, disclosed aremethods of treating cancer comprising administering to a subject aneffective amount of a composition comprising a plasma membrane vesiclecomprising two or more NK cell effector agents, wherein the plasmamembrane can comprise IL-15 and 41BBL. In some aspects, the plasmamembrane can further comprise IL-21. Also disclosed are methods oftreating cancer comprising administering to a subject an effectiveamount of a composition comprising a plasma membrane vesicle comprisingtwo or more NK cell effector agents, wherein the plasma membrane cancomprise IL-21 and 41BBL. Also disclosed are methods of treating cancercomprising administering to a subject an effective amount of acomposition comprising a plasma membrane vesicle comprising two or moreNK cell effector agents, wherein the plasma membrane vesicle comprisesIL-15 and IL-21.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agent, whereinthe plasma membrane vesicle can be purified from NK cell feeder cells.NK cell feeder cells can be irradiated autologous or allogeneicperipheral blood mononuclear cells (PBMCs), RPMI8866, HFWT, K562, K562cells transfected with membrane bound IL-15 and 41BBL), K562 cellstransfected with membrane bound IL-21 and 41BBL, or EBV-LCL. In someaspects, the NK cell feeder cells can be K562 cells transfected withmembrane bound IL-21 and 41BBL or K562 cells transfected with membranebound IL-15 and 41BBL.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agent, whereinan effective amount of the composition stimulates expansion of NK cells.Any of the disclosed plasma membrane vesicle compositions can be used inthese methods.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent. Aneffective amount of the composition can stimulate expansion of NK cells.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe composition comprises a membrane self-inserting peptide conjugatedto IL-15, IL-21, or 41BBL.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe membrane self-inserting peptide can be human Fc, GPI, transmembraneT cell receptor, or pHLIP.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe composition can comprise two or more membrane self-insertingpeptides conjugated to an NK cell effector agent.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells. NK cell effector agentscan be a cytokine, adhesion molecule or NK cell activating agent. Insome aspects, the NK cell effector agent can be IL-15, IL-21 or 41BBL.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells, wherein the plasmamembrane vesicle comprises IL-15 and IL-21. In some aspects, the plasmamembrane vesicle can further comprise 41BBL.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells, wherein the plasmamembrane vesicle comprises IL-15 and 41BBL. In some aspects, the plasmamembrane vesicle can comprise IL-21 and 41BBL.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells, wherein the plasmamembrane vesicle can be purified from NK cell feeder cells. NK cellfeeder cells can be irradiated autologous or allogeneic peripheral bloodmononuclear cells (PBMCs), RPMI8866, HFWT, K562, K562 cells transfectedwith membrane bound IL-15 and 41BBL), K562 cells transfected withmembrane bound IL-21 and 41BBL, or EBV-LCL. In some aspects, the NK cellfeeder cells can be K562 cells transfected with membrane bound IL-21 and41BBL or K562 cells transfected with membrane bound IL-15 and 41BBL.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells, wherein an effectiveamount of the composition stimulates expansion of NK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells, wherein administering toa cell population comprises administering the composition to a subject,wherein the subject comprises the cell population.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of the composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells. An effective amount ofthe composition can stimulate expansion of NK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of the composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells, wherein the compositioncomprises a membrane self-inserting peptide conjugated to IL-15, IL-21,or 41BBL.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of the composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells, wherein the membraneself-inserting peptide can be human Fc, GPI, transmembrane T cellreceptor, or pHLIP.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of the composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells, wherein administering toa cell population comprises administering the composition to a subject,wherein the subject comprises the cell population.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of the composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells, wherein the compositioncomprises at least two membrane self-inserting peptide conjugates,wherein the membrane self-inserting peptide conjugates are conjugated toan NK cell effector agent. At least two membrane self-inserting peptideconjugates can be conjugated to the same or different NK cell effectoragents.

Disclosed are methods of modulating the immune system comprisingadministering to a subject one or more of the compositions comprising amembrane self-inserting peptide conjugated to an NK cell effector agentor a plasma membrane vesicle comprising an NK cell effector agent.Methods of modulating the immune system can comprise reducing the numberof activated T cells, expanding the number of NK cells, or reducing thenumber of dendritic cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 is a depiction of a plasma membrane from K562-mb15-41BBL cellscontaining stimulatory ligands (IL-15 and 41BBL) either alone or loadedwith magnetic nano-particles, a C18 silica or polystyrene bead, or asilica bead.

FIG. 2 is a representation of NK cell effector agents, which include41-BBL, IL-15, IL-21, MICA, 2B4, and other. The other includes but isnot limited to BCM1/SLAMF2, IL-2, IL-12. Also shown is a genericmembrane self-inserting peptide (MBI). The NK cell effect agent and MBIcan be crosslinked together and self-insert into tumor cells.

FIG. 3 presents dot plots of CD56-PE vs. CD3-APC, showing CD56+CD3− NKcell population increases as the concentration of K562-mb21 plasmamembrane in culture increases after 10 days.

FIG. 4 presents a bar graph depicting the increase in NK cell percentagein cultured cells resulting from an increase in concentration ofK562-mb15 or K562-mb21 plasma membrane.

FIG. 5 presents bar graphs showing an increase in NK cell expansion withincreasing concentrations of PM-mb15-41BBL or PM-mb21-41BBL membranevesicles in culture.

FIG. 6 presents a bar graph showing the percent cytotoxicity of mbIL15K562, mbIL21 K562, mbIL15 PM, or mbIL21 PM against K562, KG1, and HL-60cells at day 10 of culture. NK cells expanded with plasma membrane fromK562mb15 and K562mb21 elicit comparable cytotoxic responses against AML(KG1 and HL-60) and CML (K562) cell lines when compared to live feedercell cultured NK cells.

FIG. 7 presents histograms showing IL-15-FITC and 41BBL-PE. Latex or C18Silica beads were coated with K562mb15 plasma membrane containingmembrane-bound IL15 and 41BBL. Both latex and C18 Silica beads showedthe presence of IL-15 and 41BBL after coating. Isotype is the lightercolored line.

FIGS. 8A and 8B depict debris found in cultures after NK cell expansion.a) FSC vs. SSC of PBMC (red) together with plasma membrane at day 10 ofculture. b) FSC vs. SSC of PBMC (red) together with K562mb15 feeder celldebris (green). The top and bottom figures represent separate cultureson day 10. The top figure has significantly more feeder cell debris vs.the bottom figure.

FIG. 9 shows that PM-mb15-41BBL vesicles efficiently expand NK cells.PBMC were co-cultured with K562-mb15-41BBL feeders or were stimulatedwith PM-mb15-41BBL vesicles (at 100 or 200 μg of membrane protein/ml)for 21 days. The cell content was tested every 2-3 days and media wereexchanged as needed. The figure depicts one representative expansion out3 performed with PBMCs derived from 3 different donors. Each pointrepresents average of duplicate cultures.

FIG. 10 shows that PM-mb15-41BBL vesicles selectively expand NK cells.PBMC were stimulated with PM-mb15-41BBL vesicles at 200 μg of membraneprotein/ml for 14 days. The cell count and content was tested every 2-3days by staining the cells with anti-CD56-PE and anti-CD3-APC antibodiesand analyzing the content on an Accuri flow cytometer. In allexperiments performed to date CD56+CD3− NK cells selectively expandedwithin PBMCs when stimulated with PM-mb15-41BBL vesicles and by day 14thconsisted >95% of the cellular content. The figure depicts onerepresentative expansion out of 3 performed with PBMCs derived from 3different donors.

FIG. 11 shows that NK cell content increases with the concentration ofPM-mb15-41BBL vesicles. PBMC were stimulated with varying amounts ofplasma membrane vesicles for 14 days. Cell content measured usinganti-CD56-PE and anti-CD3-APC. Each point represents average ofduplicate cultures.

FIG. 12 shows that optimized PM-mb15-41BBL vesicles expand NK cellsequally well as live feeder cells. PBMC were co-cultured with optimized(n=6) or initial (n=4) PM vesicle preparations at 200 μg of membraneprotein/mL or were co-cultured with 1×10⁶/mL K562-mb15-41BBL livefeeders (n=6, Feeders positive ctrl) or 50 ng/mL soluble IL-15 and 50ng/mL soluble 41BBL (n=6, SC Ctrl) for up to 20 days. The plot depictsthe fold of NK cell expansion after 12-13 days in culture.

FIG. 13 shows the growth rate of PM-mb15-41BBL vesicles expanded NKcells slows down after two weeks in culture. PBMC were co-cultured withPM vesicles at 200 μg of membrane protein/mL for over 4 weeks. ½ ofmedia was replaced every other day after day 3. 100 μg of PM vesicleswas replaced with the media replacement every other day. Cell contentmeasured using anti-CD56-PE and anti-CD3-APC. Each point representsaverage of duplicate cultures. The figure depicts one representativeexpansion out of 3 performed with PBMCs derived from 3 different donors.

FIG. 14 shows PM-mb15-41BBL vesicles expanded NK cells are cytotoxicagainst CML and AML targets. Cytotoxicity assay performed on day 13-14using PanToxiLux kit measuring Caspase/Granzyme activity in labeledtarget cells. Target cells at 0.5×10⁶/mL were co-incubates with expandedNK cells at varying ratios in duplicate for 1.5 hours and then analyzedby flow cytometry. The amount of spontaneous target cell death wasdetermined using a “Target Alone” control. Percent cytotoxicity wasdeterment by subtracting spontaneous cell death from target cell deathand then dividing by total target cells. Figure depicts onerepresentative cytotox assay out of 3 performed with PBMCs derived from3 different donors.

FIG. 15 shows PM-mb15-41BBL vesicles expanded NK target cells ascompared to freshly isolated NK cells. Initial cytotoxicity assayperformed on freshly isolated NK cells from healthy donors. NK'sisolated from PBMC after Ficoll using Stem Cell Technologies NegativeSelection kit were allowed to rest and pre-activate overnight in growthmedia+FBS+high (1000 U/mL) IL2 before testing cytotoxicity. NK'sexpanded from PBMC with PM vesicles at 200 μg of membrane protein/mL for13-14 days were pre-activated with high (1000 U/mL) IL2 overnight beforetesting cytotoxicity. The figure depicts one representative expansionout of 3 performed with PBMCs derived from 3 different donors.

FIG. 16 shows PM-mb15-41BBL vesicles expanded NK cells are cytotoxicagainst patient primary AML cells. Cytotoxicity of NK cells culturedwith PM vesicles at 200 μg of membrane protein/mL for 13-14 days weretested against primary tumor from 2 different patients. Tumor cells werecollected from bone marrow or peripheral blood of patients that signedinformed consent. Leukemia blasts were separated by Ficoll gradient andcryopreserved for future experiments. Thawed tumor samples wereincubated in media overnight to recover before using in cytotox assays.Target tumor cells were stained with TFL4 dye to distinguish fromeffectors. Additionally, tumor cells were stained with anti-CD34-PE todistinguish patient tumor cells from healthy patient PBMC. The figuredepicts one representative expansion out of 3 performed with PBMCsderived from 3 different donors.

FIG. 17 shows PM-mb15-41BBL vesicles expand NK cells in vivo in NSGmice. PBMC derived from two different donor were stimulated withPM-mb15-41BBL vesicles at 200 μg of membrane protein/ml for 7 days. Onday seven cells were counted and analyzed for cell content, spun down,resuspended at 15 mln NK cells/mL in 100 μL of clear RPMI with 50 U/mLof IL 2 and injected into the tail vain of NSG mouse. Mice were injected3 times a week with PM-mb15-41BBL vesicles (200 μg of membraneprotein/ml) and low 100 U or 1000 U dose of IL-2. On day 1 and 4 postinjection 50-100 μL of mouse blood was drawn and analyzed for presenceand number of human CD45+ lymphocytes, CD3−CD56+ NK cells and CD3+CD56−T cells. The graph represents fold expansion of NK cells between days1-3 post injection.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

A significant portion of donor lymphocyte infusion mediatedgraft-versus-tumor (GVT) effect may be due to natural killer (NK) cells.The infusion of NK cells isolated from donor blood could producebeneficial GVT effects without causing GVHD. Preclinical and clinicaldata has shown effectiveness of NK cell infusions leading towardcomplete remission without any GVHD. Thus, NK cell infusion, incombination with autologous transplantation, or as a standalonetreatment, offers an innovative, and potentially very effective,alternative for those patients who do not have a matched donor,experience relapse, or do not qualify for transplant.

Infusions of NK cells are a treatment option for patients with cancerssusceptible to NK cell lysis, including blood cancers (such as acutemyeloid leukemia or multiple myeloma) and several solid tumors (e.g.brain tumor, Ewing sarcoma and rhabdomyosarcoma) (Harada, Saijo et al.2002; Ruggeri, Capanni et al. 2002; Miller, Soignier et al. 2005; Cho,Shook et al. 2010). Increased numbers of functional NK cells can alsosignificantly enhance the efficacy of therapeutic antibodies used intreatment of several cancers, including lymphomas, colorectal cancer,lung cancer, and breast cancer, among others (Hatjiharissi, Xu et al.2007; Triulzi, Vertuani et al. 2010; Houot, Kohrt et al. 2011; Tai,Horton et al. 2012). These types of personalized treatments are,however, very costly, with a typical antibody-containing regimen costingtens of thousands of dollars. Furthermore, the expected efficacy ofexisting methods is often not achieved due to the lack of immune cellengagement in immune compromised cancer patients (Dewan, Takada et al.2009; Mamessier, Sylvain et al. 2011).

To be effective as a cancer treatment method, it is desirable to achievea degree of NK cell expansion that reaches an effective therapeuticdose. Several studies have shown that NK cells proliferate in an invitro culture exponentially and preferentially within a mixture ofperipheral blood mononuclear cells (PBMC) when stimulated with acombination of cytokines (such as IL-15 or IL-21) and ligands foractivating receptors (such as 4-1BBL) expressed on the surface ofstimulator cells (Imai, Iwamoto et al. 2005; Cho and Campana 2009; Lee,Verneris et al. 2010; Somanchi, Senyukov et al. 2011).

For cytokines IL-15 and IL-21, cross-presentation of membrane boundinterleukin, as in normal dendritic cells, induces expansion of NK cellsmore potently than the soluble form of these cytokines. Moreover, undersuch stimulation conditions, only a low concentration of soluble IL-2 isrequired for NK cell survival, thus allowing for selective expansion ofNK cells within a PBMC mixture without observable proliferation of Tcells. The soluble form of IL-15 and IL-21 cytokines or high dose IL-2stimulate more potently the proliferation of T cells than of NK cells. Apreviously published study by Campana and coworkers has shown that in anin vitro culture stimulation of NK cells with the K562 cell line havingmembrane bound IL-15 and 4-1BBL leads to a potent expansion of NK cellsthat is not observed with K562 cells expressing either of the moleculesalone (Imai, Iwamoto et al. 2005; Fujisaki, Kakuda et al. 2009).However, NK cell expansion was limited to several divisions and thecells achieved senescence and stopped proliferating, coinciding with theobservation of telomere shortening. In a follow-up study, stimulationwith membrane bound IL-21 instead of IL-15 was found to stimulatecontinuous propagation of NK cells over countless generations allowingfor continuous expansion of NK cells provided that the culture isperiodically replenished with fresh stimulatory cells (Somanchi,Senyukov et al. 2011; Denman, Senyukov et al. 2012). While these methodsallow for efficient in vitro NK cell expansion, the need for live feedercells makes the methodology difficult to transfer to clinical settings.Also, NK cells that are infused into the patient will likely stopdividing due to the lack of continued stimulation by the feeders.Furthermore, there is still a lack of information about the ability ofin vitro cultured NK cells to function as intended when re-infused intoa patient (Miller 2009).

Currently IL-2 administration is the only FDA approved method ofexpansion of NK cells in vivo. IL-15 is currently being tested in aPhase I clinical trial as an alternative approach to IL-2 administrationbut based on preclinical findings it is still expected to havesignificant toxicity if administered systematically. Thus, both methodscarry significant toxicities to patients and also induce proliferationof T-cells including regulatory T-cells leading to short persistence (onaverage less than 21 days) of NK cells.

A successful pilot trial showed that infusion of purified NK cellsisolated from donor's blood is safe and can lead to complete remissionof AML, with no GVHD. To reach the therapeutic dose, NK cells wereexpanded in vivo in lymphodepleted patients by daily administration ofhigh dose IL-2. However, the intensive conditioning regimen required forlymphodepletion and the high doses of IL-2 used in this study resultedin significant toxicity and prolonged hospitalization, and in manycases, low in vivo expansion on NK cells. Moreover, systemicadministration of IL-2 leads to proliferation of regulatory T cells thatsuppress the numbers and function of NK cells, thereby limiting theirpersistence and efficiency in the patient. Thus, alternative approachesfor in vivo or ex vivo expansion of NK cells are needed.

The efficacy of NK cell immunotherapy is dependent on the dose of NKcells administered to the patient or reached after infusion through invivo expansion. Currently available techniques are limited by theirinability to achieve the level of NK cell expansion required to achievea therapeutic effect in a patient. The lack of a clinical expansionprotocol is a major barrier to the progress of NK cell-basedimmunotherapy. Current ex vivo expansion protocols use a combination ofhigh dose cytokines with activating ligands expressed onleukemia-derived feeder/stimulator cell lines, posing a significantdownside for transfer to clinical settings in most centers and are notamenable for direct in vivo expansion. The use of particle technologydescribed herein eliminates the need for stimulator cells, thussimplifying the methodology and allowing direct and selective in vivoexpansion.

Several groups have pursued a method to expand NK cells ex vivo.However, most of the currently developed ex vivo methods rely onco-culture systems of tumor cell lines and NK cells in the presence ofhigh concentrations of various cytokines, mostly IL-2 (Reviewed in Choand Campana 2009; Suck and Koh 2010). Cells used to trigger NK cellproliferation include irradiated autologous or allogeneic PBMCs,RPMI8866, HFWT, K562, K562-mb15-41BBL (K562 transfected with 4-1BBL andmembrane-bound IL-15), K562-mb21-41BBL and EBV-LCL(Harada, Saijo et al.2004; Imai, Iwamoto et al. 2005; Berg, Lundqvist et al. 2009; Fujisaki,Kakuda et al. 2009; Siegler, Meyer-Monard et al. 2010). Althoughexpansion of NK cells can be significant with some of these cell lines(30-10,000 fold within 7-21 days), the use of feeder cells posessignificant downsides for transfer into a clinical setting in mostcenters due to the requirement for a current Good Manufacturing Practice(cGMP) facility, which costs several million dollars (Reviewed in Choand Campana 2009; Suck and Koh 2010). Furthermore, continuous culturingof feeder cells is costly and requires support of dedicated personnel.The National Institutes of Health (NIH) currently provides support inthe manufacturing of cells for cellular therapy in the form ofProduction Assistance for Cellular Therapy (PACT). However, NK cellsappear to lose their activity during cryopreservation (PACT workshoppresentation). Thus, the storage and transport of expanded NK cells fromthe site of production to the transplant center is another obstacle insuccessful application of the therapy. An additional concern is thepotential for infusion of live feeder cells and/or genetic materialreleased from those transformed cells and culture components (e.g. fetalbovine serum) into a recipient patient.

Miltenyi Biotech has introduced an in vitro expansion kit that usesantibody-coated beads to crosslink activating NK cell receptors.However, this method requires the use of high concentration IL-2. Whileuseful for laboratory applications, this method cannot be transferred toclinical settings because NK cells cultured using high concentrations ofcytokines undergo rapid apoptosis after infusion due to cytokinewithdrawal (Miller 2009).

Expansion of NK cells within PBMC has been reported with a highconcentration of IL-2 and stimulation with anti-CD3 antibody for thefirst five days (Carlens, Gilljam et al. 2001; Alici, Sutlu et al.2008). The overall NK cell expansion was close to 1000-fold, but most ofthe NK cells were actually NK-like T cells (Berg and Childs 2010). Thus,all of the methods pose significant difficulties for the transfer toclinical applications and none of the methods can be used in direct invivo expansion.

A. Plasma Membrane Vesicles

Disclosed are plasma membrane vesicles comprising at least one NK celleffector agent. NK cell effector agents can be a cytokine, an adhesionmolecule, or an NK cell activating agent (i.e. stimulatory ligand).Examples of cytokines can be, but are not limited to, IL-2, IL-12,IL-15, IL-21, and IL-18. Examples of adhesion molecules can be, but arenot limited to LFA-1, MICA, BCM/SLAMF2. Examples of NK cell activatingagents can be, but are not limited to, 41BBL and BCM/SLAMF2. In someaspects, a member of one group can also be a member of another group.For example, BCM/SLAMF2 can be both an adhesion molecule and NK cellactivating agent. The NK cell effector agent can be but is not limitedto, 41BBL, IL-2, IL-12, IL-15, IL-21, IL-18, MICA, LFA-1, 2B4, andBCM/SLAMF2. Plasma membrane vesicles are vehicles used to carry NK celleffector agents. The NK cell effector agents can be membrane bound.While the NK cell effector agents are membrane bound, other therapeuticor diagnostic agents can be transported in the interior of the plasmamembrane vesicle.

In some aspects, the plasma membrane vesicle comprises at least onemembrane bound cytokine. Thus, disclosed are plasma membrane vesicles,wherein the plasma membrane vesicle comprises an NK cell effector agent,wherein the NK cell effector agent is IL-15, IL-21 or 41BBL.

Disclosed are compositions comprising a plasma membrane vesicles,wherein the plasma membrane vesicle comprises two or more NK celleffector agents. In some aspects, at least one NK cell effector agentcan be a cytokine. For example, the plasma membrane can comprise IL-15and 41BBL; IL-15, 41BBL and IL-21; or IL-21 and 41BBL. In some aspects,the plasma membrane vesicle comprises at least two membrane boundcytokines. For example, a plasma membrane vesicle can comprise membranebound IL-15 and membrane bound IL-21.

The plasma membrane vesicles can be used alone, loaded with magneticnanoparticles or mounted on a solid surface such as but not limited to asilica or latex bead. In some aspects, plasma membrane vesicles cansurround or coat a microparticle. Plasma membrane coated microparticlescan be made with any of the plasma membrane vesicles described herein.For example, disclosed are microparticles coated with a plasma membranecomprising IL-15 and 41BBL or IL-21 and 41BBL. The plasma membranecoated microparticles can be loaded with magnetic particles, silicabeads, polystyrene beads, latex beads, contrasting agents, or knowntherapeutics, such as cancer therapeutics.

Disclosed are compositions comprising two or more plasma membranevesicles, wherein the plasma membrane vesicles comprise an NK celleffector agent. For example, compositions can comprise a plasma membranevesicle that contains IL-15 and 41BBL and a plasma membrane vesicle thatcontains IL-21 and 41BBL.

Disclosed are compositions comprising at least one compositioncomprising a membrane self-inserting peptide conjugated to an NK celleffector agent and at least one composition comprising a plasma membranevesicle, wherein the plasma membrane vesicle comprise an NK celleffector agent. The NK cell effector agent conjugated to the membraneself-inserting peptide can be the same or different from the NK celleffector agent present in the plasma membrane vesicle.

Plasma membrane vesicles are vesicles made from the plasma membrane of acell or artificially made (i.e. liposomes). Plasma membrane vesicles canbe prepared using any of the techniques known in the art. For example,common plasma membrane preparation protocols can be used or commonprotocols for preparing liposomes. The plasma membrane vesicle cancontain a lipid bilayer or simply a single layer of lipids. Artificiallymade plasma membranes, such as liposomes, can be prepared using a lipidbilayer and membrane self-inserting peptide conjugates as describedbelow.

B. Membrane Self-Inserting Peptides Conjugates

Disclosed are compositions comprising a membrane self-inserting peptideconjugated to an NK cell effector agent. These compositions can also bereferred to as membrane self-inserting peptide conjugates.

NK cell effector agents can be a cytokine, an adhesion molecule, or anNK cell activating agent (i.e. stimulatory ligand). Examples ofcytokines can be, but are not limited to, IL-2, IL-12, IL-15, IL-21, andIL-18. Examples of adhesion molecules can be, but are not limited toLFA-1, MICA, BCM/SLAMF2. Examples of NK cell activating agents can be,but are not limited to, 41BBL and BCM/SLAMF2. In some aspects, a memberof one group can also be a member of another group. For example,BCM/SLAMF2 can be both an adhesion molecule and NK cell activatingagent. The NK cell effector agent can be but is not limited to, 41BBL,IL-2, IL-12, IL-15, IL-21, IL-18, MICA, LFA-1, 2B4, and BCM/SLAMF2.Thus, disclosed are compositions comprising a membrane self-insertingpeptide conjugated to an NK cell effector agent, wherein the NK celleffector agent can be IL-15, IL-21 or 41BBL.

Disclosed are compositions comprising a membrane self-inserting peptideconjugated to an NK cell effector agent, wherein the membraneself-inserting peptide is a molecule that promotes insertion into amembrane and can be human Fc, GPI, transmembrane T cell receptor, orpHLIP. The membrane self-inserting peptide can be any peptide known toinsert into a cell membrane. Depending on the use of the membraneself-inserting peptide conjugate, certain membrane self-insertingpeptides can be better choices than others. One of skill in the artwould understand what membrane self-inserting peptide is ideal underdifferent circumstances. For example, for in vivo use, pHLIP membraneself-inserting peptide can be used. pHLIP membrane self-insertingpeptides insert into the membrane only under conditions of low pH.Therefore, pHLIP conjugates will not insert into cell membranes undernormal physiological conditions. However, upon injection into a tumorenvironment, the pHLIP conjugate can insert into the cell membrane oftumor cells because the tumor environment is more acidic than normalphysiological conditions. This insertion into the tumor environmentallows for activation of NK cells in the area of the tumor. Using pHLIPthus prevents unwanted insertion into random cell membranes.

Disclosed are compositions comprising two or more membraneself-inserting peptide conjugates, wherein the membrane self-insertingconjugates are conjugated to an NK cell effector agent. Any of thedisclosed membrane self-inserting conjugates described herein can beused in these compositions. The two or more membrane self-insertingconjugates can have the same or different NK cell effector agent.

The membrane self-inserting peptides can be conjugated to an NK celleffector agent in a variety of ways. Techniques for conjugating are wellknown in the art. In some aspects, the membrane self-inserting peptideconjugates can be fusion proteins. Fusion-proteins can be produced inbacterial cells. The fusion proteins can consist of the NK cell effectoragent conjugated to a lipophilic molecule such as hydrophobic peptide,GPI, or human Fc for anchoring into liposomes or cellular membranes(Hunt, Rath et al. 1997; Kueng, Leb et al. 2007; Paulick, Forstner etal. 2007; Paulick, Wise et al. 2007; Reshetnyak, Segala et al. 2007).cDNA vectors for these fusion proteins can be ligated into an expressionplasmid, which allows expression in bacterial (E. coli), insect, ormammalian cells. The cDNA vector can be FLAG- or HIS-tagged. Bacterialcells can be transfected using standard CaCl transfection methods, suchas that described in Sambrook et al., Molecular Cloning: A LaboratoryManual. 2nd ed. Cold Spring Harbor Laboratory Press (1989). Bacterialcells can be cultured in LB media and cells can be harvested and lysedusing a French Press. Proteins of interest can be purified from lysatesby affinity chromatography. Palmitate-conjugated protein A and purifiedFc fusion proteins can be conjugated as described in the literature bymixing 1:2 (w/w) at 4 degrees C. (see Kim & Peacock, Journal ofImmunological Methods, 1993 Jan. 14; 158(1):57-65 and Liu et al.,Journal of Immunology, 2007 Mar. 1; 178(5); 3301-3306). The conjugatescan then be directly injected intratumorally or can be incorporated intoliposomes.

Membrane self-inserting peptide conjugates can be incorporated intoliposomes. For example, GPI can be coupled with 41BBL, IL-2, IL-12,IL-15, IL-21, MICA, 2B4, or BCM/SLAMF1 and can be incorporated intoliposomes.

Liposomes can be prepared by dissolving lipids in chloroform andevaporating under nitrogen to form a film. Small unilamellar liposomescan be formed by resuspending lipids in water and sonicating with a bathsonicator. Either protein A/Fc fusion protein conjugates or GPI coupledproteins can be mixed with liposomes. Protein insertion can be such thatthe protein A or GPI anchor can insert into the lipid bilayer, leavingthe functional stimulatory protein on the extracellular region.Liposomes can then be PEGylated to increase lifetime. Protein-coatedliposomes can then be used for cancer therapy by ex vivo expansion ofimmune effector cells or by direct intravenous injection to promote invivo expansion/activation of immune cells.

Types of conjugation and methods for conjugating are known to the art.The term “conjugate” refers to the membrane self-inserting peptide beingconjugated, coupled, or linked to another composition such as a peptideor protein. For example, membrane self-inserting peptide conjugates canbe fusion proteins wherein the membrane self-inserting peptide isconjugated to another protein via a disulfide bond. Coupling orconjugating can mean that there is a chemical linkage between themembrane self-inserting peptide and the NK cell effector agent.

In some aspects, the NK cell effector agent can be conjugated tomembrane self-inserting peptides or GPI anchors for in situself-assembly. For example, 41-BBL and IL-21 can be conjugated into apHLIP peptide which inserts itself into cellular membranes under acidicconditions, thereby allowing the anchoring of the stimulatory ligandsinto cells in the proximity of tumor (Reshetnyak, et al. 2006; Andreev,et al. 2007; Reshetnyak, et al. 2007). The NK cell effector agents41BBL, IL-2, 11-12, IL-15, IL-21, and BCM/SLAMF2 can be produced inbacterial cells or purchased from commercially available sources. cDNAvectors for these proteins can be ligated into pTriEX expression plasmidwhich allows expression in bacterial (E. coli), insect, or mammaliancells. The cDNA vector can code for expression of FLAG- or HIS-tag.Bacterial cells can be transfected using standard CaCl transfectionmethods. Bacterial cells can be cultured on LB media. Cells can beharvested and lysed using a French press. Proteins of interest can thenbe purified from lysates by affinity chromatography.

pHLIP can be prepared by solid-phase peptide synthesis using9-fluorenylmethyloxycarbonyl chemistry and the product can be purifiedon a C18 column by reverse-phase chromatography. pHLIP can then beconjugated to stimulatory human protein ligands by incubating with acrosslinker, such as benzophenone-4-iodoacetamide. After several washes,the conjugated pHLIP protein can be resuspended in media (saline, forexample) and injected intratumorally or intravenously. Based on evidencefrom prior literature (Imai, Iwamoto et al. 2005; Liu, Breiter et al.2007; Fujisaki, Kakuda et al. 2009; Somanchi, Senyukov et al. 2011;Denman, Senyukov et al. 2012) and presented experimental results,interaction of NK cells with stimulatory ligands such as IL-21 or IL-15and 41-BBL on the surface of such modified tumor cells can stimulate insitu NK cell expansion and trigger their cytotoxic response toward atumor. This type of stimulatory approach can be used for treatments ofsolid tumors such as ovarian cancer where NK stimulatory ligands thatinsert in situ into tumor cells under acidic pH can be injected intointraperitoneal space of patients with low dose IL-2 alone or togetherwith NK cells (Geller, Cooley et al. 2011). There is strong evidencethat cytotoxic lymphocytes that express high levels of FCγIII R (CD16)such as NK cells are crucial for the efficacy of cancer therapy withtherapeutic antibodies(Kute, Savage et al. 2009; Reim, Dombrowski et al.2009; Mamessier, Sylvain et al. 2011). Thus, this approach can also beused in combination with therapeutic antibodies.

C. Combination of Plasma Membrane Vesicles and Membrane Self-InsertingPeptide Conjugates

Disclosed are compositions comprising one or more of the disclosedplasma membrane vesicles and one or more of the disclosed membraneself-inserting peptide conjugates. The plasma membrane vesicle and themembrane self-inserting peptide conjugate can comprise the same NK celleffector agent or different NK cell effector agents.

D. Methods of Expanding Natural Killer Cells

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the cell population comprises NK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells.

Also disclosed are methods of expanding NK cells comprisingadministering a composition that comprises one of the disclosed plasmamembrane vesicles and a composition comprising one of the disclosedmembrane self-inserting peptide conjugates. In some aspects, onecomposition can be formulated to contain one or more plasma membranevesicles and one or more membrane self-inserting peptide conjugates.

Expanded NK cells and/or compositions used to expand NK cells can beused as a treatment method for patients having cancers that aresusceptible to NK cell mediated lysis as well as for patients who haveundergone hematopoietic stem cell transplant. NK cell expandingcompositions can be used to increase the amount of cytotoxic NK cellsafter stem cell transplant for increased clearance of residual tumorcells and/or for relapse prevention. The NK cell-expanding compositionscan also be used to treat patients with viral infection.

NK cell expanding compositions can be used as a post NK cell infusiontreatment method to increase the numbers and in vivo persistence ofcytotoxic NK cells for increased efficacy of NK cell therapy (i.e.number of patients that achieve remission and/or remain in remission).

NK cells with or without NK cell-expanding compositions will be used incombination with therapeutic antibodies for treatment of various cancersincluding, but not limited to, lymphomas, colorectal, lung, colon, headand neck, and breast cancers to increase the number of patients thatrespond to the therapeutic antibody therapy and achieve remission and/orremain in remission.

1. Expanding NK Cells with Plasma Membrane Vesicles

Plasma membrane vesicles comprising at least one NK cell effector agentcan be used to expand NK cells in vitro or in vivo. The methods ofexpanding NK cells can comprise administering to a cell population aneffective amount of a composition comprising a plasma membrane vesiclecomprising at least one NK cell effector agent.

NK cell effector agents can be a cytokine, adhesion molecule or NK cellactivating agent. Disclosed are methods of expanding NK cells comprisingadministering to a cell population an effective amount of a compositioncomprising a plasma membrane vesicle comprising at least one NK celleffector agent, wherein the cell population comprises NK cells, whereinthe NK cell effector agent can be IL-15, IL-21 or 41BBL. In someaspects, the plasma membrane vesicle can comprise any combination of NKcell effector agents. For example, a plasma membrane vesicle cancomprise IL-15 and IL-21; IL-15 and 41BBL; IL-21 and 41BBL; or IL-15,IL-21 and 41BBL. Thus, a plasma membrane vesicle containing anycombination of one or more of IL-15, IL-21 and 41BBL can be used toexpand NK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein the plasma membrane vesicle can be purified from NK cell feedercells. The NK cell feeder cells can be irradiated autologous orallogeneic peripheral blood mononuclear cells (PBMCs), RPMI8866, HFWT,K562, K562 cells transfected with membrane bound IL-15 and 41BBL), K562cells transfected with membrane bound IL-21 and 41BBL, or EBV-LCL. Insome aspects, the NK cell feeder cells can be K562 cells transfectedwith membrane bound IL-21 and 41BBL or K562 cells transfected withmembrane bound IL-15 and 41BBL.

In some aspects, the plasma membrane vesicles used in the methods toexpand NK cells can be derived from any cell type. The plasma membraneof any cell can be altered to contain the NK cell effector agents ofinterest.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein an effective amount of the composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agentstimulates expansion of NK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising aplasma membrane vesicle comprising at least one NK cell effector agent,wherein administering to a cell population comprises administering thecomposition to a subject, wherein the subject comprises the cellpopulation.

In some aspects of the methods, cytotoxic NK cells selectively expand invivo in the presence of a combination of NK cell effector agentsincluding but not limited to membrane bound cytokines, adhesionmolecules and NK cell activating agents. The NK cell effector agents canbe delivered in a form of an empty or loaded cell membrane vesicle,liposome, cell membrane coated bead or lipid coated bead (FIG. 1 ). Themolecules that can be loaded into the plasma membrane vesicles include,but are not limited to, IL-2 for local delivery to the site of NK-cellexpansion, contrasting agents for detection to monitor bio-distributionand clearance, or super-magnetic particles to deliver the particles tospecific locations e.g. lymph nodes or tumors. The magnetic particlescan also allow quick removal and detection.

2. Expanding NK Cells with Membrane Self-Inserting Peptide Conjugates

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising atleast one membrane self-inserting peptides conjugated to an NK celleffector agent, wherein the cell population comprises NK cells. Membraneself-inserting peptides conjugated to an NK cell effector agent can beused to expand NK cells. Membrane self-inserting peptide conjugates canbe administered as a peptide conjugate or they can be used to make acomposition such as liposomes containing the membrane self-insertingpeptide conjugates and then the liposomes can be administered.

Disclosed are methods of expanding NK cells comprising administering toa subject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinan effective amount of the composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agentstimulates expansion of NK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein an effective amount of the composition stimulates expansion ofNK cells.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the composition can comprise a membrane self-inserting peptideconjugated to IL-15. In some aspects, the composition can comprise amembrane self-inserting peptide conjugated to IL-21. In some aspects,the composition can comprise a membrane self-inserting peptideconjugated to 41BBL. In some aspects the NK cell effector agent is anycytokine, adhesion molecule or NK cell activating agent.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the membrane self-inserting peptide can be human Fc, GPI,transmembrane T cell receptor, or pHLIP.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agentwherein administering to a cell population comprises administering thecomposition to a subject, wherein the subject comprises the cellpopulation.

Disclosed are methods of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the composition comprises at least two membrane self-insertingpeptide conjugates, wherein the membrane self-inserting peptideconjugates are conjugated to an NK cell effector agent. The at least twomembrane self-inserting peptide conjugates can be conjugated to the sameor different NK cell effector agents. For example, the methods ofexpanding NK cells can include administering a composition thatcomprises a membrane self-inserting peptide conjugated to a cytokine anda membrane-self-inserting peptide conjugated to an NK cell activatingagent. In some aspects, methods of expanding NK cells can compriseadministering two or more separate compositions wherein each compositiononly contains one membrane self-inserting peptide conjugate.

3. NK Cells

Human NK cells are a subset of peripheral blood lymphocytes defined bythe expression of CD56 or CD16 and the absence of T cell receptor (CD3)(Ljunggren and Malmberg 2007; Woan and Reddy 2007). NK cells sense andkill target cells that lack major histocompatibility complex (MHC)-classI molecules. NK cell activating receptors include, among others, thenatural cytotoxicity receptors (NKp30, NKp44 and NKp46), and lectin-likereceptors NKG2D and DNAM-1. Their ligands are expressed on stressed,transformed, or infected cells but not on normal cells, making normalcells resistant to NK cell killing (Bottino, Castriconi et al. 2005;Gasser, Orsulic et al. 2005; Lanier 2005). NK cell activation isnegatively regulated via inhibitory receptors, such as killerimmunoglobin (Ig)-like receptors (KIRs), NKG2A/CD94, and leukocyteIg-like receptor-1 (LIR-1). Engagement of one inhibitory receptor may besufficient to prevent target lysis (Bryceson, Ljunggren et al. 2009).Hence NK cells efficiently target cells that express many stress-inducedligands, and few MHC class I ligands.

NK cells efficiently destroy tumor cells, stressed cells, and virallyinfected cells by a variety of different methods. The first is bydirectly engaging target cells, permeating their membranes, and theninjecting a protein that cleaves and activates several apoptoticproteins, thereby initiating programmed cell death (apoptosis) of thetargeted cell. The surface of an NK cell also contains protein ligandsthat can bind and activate receptors, such as the receptor fortumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL),on target cells that turn on internal signals for apoptotic programmedcell death. When stimulated, NK cells can also secrete cytokines such asINFγ and TNFα that not only inhibit viruses and tumors, but also signalinvasion to other immune cells. This broad and multimodal anti-canceractivity of NK cells make them of great interest to the medical field.

Because NK cells have a prominent role in the immune system, the abilityto expand NK cells provides treatment opportunities that were notpossible or less effective with low numbers of NK cells.

4. Expanding NK Cells Provides Other Options for the NK Cells

The methods of expanding NK cells are beneficial for treating cancer,treating viral infections, studying NK cells, treating multiplesclerosis, immune surveillance, and treating graft versus host disease.Any NK cell related disorder can be treated or affected by the expansionof NK cells. For example, diseases such as multiple sclerosis that areknown for having an increase in activated T cells can be treated withthe disclosed compositions because these compositions cause an expansionof NK cells that target and kill activated T cells. Thus, the disclosedcompositions can be used to decrease activated T cells.

E. Methods of Treating Cancer

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agent.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent.

Treating cancer with a plasma membrane vesicle comprising at least oneNK cell effector agent or a membrane self-inserting peptide conjugatedto an NK cell effector agent can occur due to the expansion of NK cellsin the presence of these compositions. The expansion of NK cells leadsto more NK cells able to target and kill tumor cells, thus reducingtumor cells and ultimately treating cancer.

The plasma membrane vesicle comprising at least one NK cell effectoragent or membrane self-inserting peptide conjugated to an NK celleffector agent can provide a preventative effect. NK cells are known toprovide immunosurveillance. Therefore, administering a composition thatresults in expansion of NK cells allows for more NK cells to provideimmunosurveillance and to target and kill pre-cancerous cells beforecancer occurs.

In some aspects, administering to a subject can include administeringthe disclosed compositions to a cell population in vitro and thenadministering those treated cells to a subject.

1. Treating with Plasma Membrane Vesicles

Plasma membrane vesicles comprising at least one NK cell effector agentcan be used to treat cancer. The methods of treating cancer can compriseadministering to a subject an effective amount of a compositioncomprising a plasma membrane vesicle comprising at least one NK celleffector agent.

NK cell effector agents can be a cytokine, adhesion molecule or NK cellactivating agent. Disclosed are methods of treating cancer comprisingadministering to a subject an effective amount of a compositioncomprising a plasma membrane vesicle comprising at least one NK celleffector agent, wherein the NK cell effector agent can be IL-15, IL-21or 41BBL. In some aspects, the plasma membrane vesicle can comprise anycombination of NK cell effector agents. The plasma membrane can comprisetwo or more NK cell effector agents. In some aspects, at least one NKcell effector agent can be a cytokine. For example, a plasma membranevesicle can comprise IL-15 and IL-21; IL-15 and 41BBL; IL-21 and 41BBL;or IL-15, IL-21 and 41BBL. Thus, a plasma membrane vesicle containingany combination of one or more of IL-15, IL-21 and 41BBL can be used totreat cancer.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agent, whereinthe plasma membrane vesicle can be purified from NK cell feeder cells.The NK cell feeder cells can be irradiated autologous or allogeneicperipheral blood mononuclear cells (PBMCs), RPMI8866, HFWT, K562, K562cells transfected with membrane bound IL-15 and 41BBL), K562 cellstransfected with membrane bound IL-21 and 41BBL, or EBV-LCL. In someaspects, the NK cell feeder cells can be K562 cells transfected withmembrane bound IL-21 and 41BBL or K562 cells transfected with membranebound IL-15 and 41BBL.

In some aspects, the plasma membrane vesicles used in the methods totreat cancer can be derived from any cell type. The plasma membrane ofany cell can be altered to contain the NK cell effector agents ofinterest.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a plasmamembrane vesicle comprising at least one NK cell effector agent, whereinan effective amount of the composition comprising a plasma membranevesicle comprising at least one NK cell effector agent stimulatesexpansion of NK cells.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising at least twoplasma membrane vesicles, wherein the plasma membrane vesicle comprisesat least one NK cell effector agent. The at least two plasma membranevesicles can comprise the same or different NK cell effector agents. Forexample, the methods of treating cancer can include administering acomposition that comprises a plasma membrane vesicle comprising amembrane bound cytokine and a plasma membrane vesicle comprising amembrane bound NK cell activating agent. In some aspects, methods oftreating cancer can comprise administering two or more separatecompositions wherein each composition only contains one plasma membranevesicle.

2. Treating with Membrane Self-Inserting Peptide Conjugates

Membrane self-inserting peptides conjugated to an NK cell effector agentcan be used to treat cancer. The membrane self-inserting peptideconjugates can be administered as a peptide conjugate or they can beused to make a composition such as liposomes containing the membraneself-inserting peptide conjugates and then the liposomes can beadministered to a subject.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinan effective amount of the composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agentstimulates expansion of NK cells.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe composition can comprise a membrane self-inserting peptideconjugated to IL-15. In some aspects, the composition can comprise amembrane self-inserting peptide conjugated to IL-21. In some aspects,the composition can comprise a membrane self-inserting peptideconjugated to 41BBL. In some aspects the NK cell effector agent is anycytokine, adhesion molecule or NK cell activating agent.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe membrane self-inserting peptide can be human Fc, GPI, transmembraneT cell receptor, or pHLIP.

Disclosed are methods of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe composition comprises at least two membrane self-inserting peptideconjugates, wherein the membrane self-inserting peptide conjugates areconjugated to an NK cell effector agent. The at least two membraneself-inserting peptide conjugates can be conjugated to the same ordifferent NK cell effector agents. For example, the methods of treatingcancer can include administering a composition that comprises a membraneself-inserting peptide conjugated to a cytokine and amembrane-self-inserting peptide conjugated to an NK cell activatingagent. In some aspects, methods of treating cancer can compriseadministering two or more separate compositions wherein each compositiononly contains one membrane self-inserting peptide conjugate.

3. Combination Treatments

The methods of treating cancer comprising administering to a subject aneffective amount of a composition comprising a plasma membrane vesiclecomprising at least one NK cell effector agent or a membraneself-inserting peptide conjugated to an NK cell effector agent can becombined with other cancer treatments. For example, the methods cancomprise administering to a subject an effective amount of a compositioncomprising a plasma membrane vesicle comprising at least one NK celleffector agent or a membrane self-inserting peptide conjugated to an NKcell effector agent and a cancer therapeutic.

In some aspects, the cancer therapeutic and the plasma membrane vesiclecan be formulated in the same composition. In some aspects, the cancertherapeutic and the plasma membrane vesicle can be formulated indifferent compositions.

The composition comprising a plasma membrane vesicle comprising at leastone NK cell effector agent and the cancer therapeutic can beadministered simultaneously or at different times.

Cancer therapeutics that can be administered in combination with theplasma membrane vesicle comprising at least one NK cell effector agentcan be any known cancer therapeutics including but not limited tochemotherapeutics and immunotherapeutics, such as but not limited toantibodies and cytokines.

F. Methods of Modulating the Immune System

Disclosed are methods of modulating the immune system comprisingadministering to a subject one or more compositions, wherein thecompositions comprise at least one plasma membrane vesicle comprising anNK cell effector agent or at least one membrane self-inserting peptideconjugated to an NK cell effector agent. In some aspects, the one ormore compositions contain a combination of plasma membrane vesicles andmembrane self-inserting peptide conjugates.

Disclosed are methods of modulating the immune system comprisingadministering to a subject one or more compositions, wherein thecompositions comprise at least one plasma membrane vesicle comprising anNK cell effector agent or at least one membrane self-inserting peptideconjugated to an NK cell effector agent, wherein modulating the immunesystem comprises reducing the number of activated T cells, expanding thenumber of NK cells, reducing the number of dendritic cells, or acombination thereof.

G. Combination Treatments

Disclosed are methods of treating cancer, viral infections, multiplesclerosis and graft-versus-host disease comprising administering to asubject one of the disclosed compositions in combination with a knowntherapeutic for the disease or disorder being treated. For example,disclosed are methods of treating cancer comprising administering to asubject a plasma membrane vesicle comprising an NK cell effector agentin combination with a known cancer therapeutic such as, but not limitedto, a chemotherapeutic, immunotherapeutic, radiation therapy or paintherapeutic.

During combination treatments, the plasma membrane vesicle or membraneself-inserting peptide conjugates disclosed herein can be administeredat the same time as the known therapeutic for the disease or disorderbeing treated. In some aspects, the plasma membrane vesicle or membraneself-inserting peptide conjugates are administered 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, or 31 days before or after the known therapeutic forthe disease or disorder being treated. In some aspects, the plasmamembrane vesicle or membrane self-inserting peptide conjugates areadministered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before orafter the known therapeutic for the disease or disorder being treated.

H. Administration

The disclosed compositions can be administered in vivo or in vivo. Insome aspects, the methods include a combination of in vitro and in vivoadministration. The compositions can be administered in vivo in apharmaceutically acceptable carrier. By “pharmaceutically acceptable” ismeant a material that is not biologically or otherwise undesirable,i.e., the material can be administered to a subject, along with theplasma membrane vesicle or membrane self-inserting peptide conjugate,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The compositions can be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, by intratumoral injection, transdermally, extracorporeally,topically or the like, including topical intranasal administration oradministration by inhalant. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the plasma membrane vesicles. Administration of thecompositions by inhalant can be through the nose or mouth via deliveryby a spraying or droplet mechanism. Delivery can also be directly to anyarea of the respiratory system (e.g., lungs) via intubation. The exactamount of the compositions required will vary from subject to subject,depending on the species, age, weight and general condition of thesubject, the severity of the disorder being treated, the particularcomposition used, its mode of administration and the like. Thus, it isnot possible to specify an exact amount for every composition. However,an appropriate amount can be determined by one of ordinary skill in theart using only routine experimentation given the teachings herein.

1. Pharmaceutically Acceptable Carriers

The compositions can be used therapeutically in combination with apharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers can be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition can be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration can be topically (includingophthalmically, vaginally, rectally, intranasally), orally, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection. The disclosedantibodies can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol vegetableoils such as olive oil, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like can be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders can be desirable.

Some of the compositions can potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

I. Devices

Disclosed are devices comprising a plasma membrane vesicle comprising anNK cell effector agent. For example, a container used during apheresiscan comprise plasma membrane vesicles comprising NK cell effectoragents. Thus, during apheresis the cells that pass through the containercan be incubated or placed into contact with the plasma membranevesicles allowing for stimulation of the NK cells and ultimately NK cellexpansion.

J. Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for preparingthe membrane self-inserting peptide conjugates, the kit comprisingmembrane self-inserting peptides. The kits also can contain reagentsused for coupling or conjugating the membrane self-inserting peptide toan NK cell effector agent.

The disclosed kits can also include NK cell effector or expansionagents. The kits can further contain components for preparing plasmamembranes or liposomes.

Nanoparticles and microparticles can be provided in the kits.

K. Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “aplasma membrane vesicle” includes a plurality of such plasma membranevesicles, reference to “the NK cell effector agent” is a reference toone or more NK cell effector agents and equivalents thereof known tothose skilled in the art, and so forth.

“Plasma membrane vesicle” refers to a preparation of a plasma membranefrom a cell or an artificially made plasma membrane or liposome.

“Membrane self-inserting peptides” are peptides that are capable ofinserting or anchoring to a cell membrane.

“Membrane self-inserting peptide conjugates” are membrane self-insertingpeptides conjugated or coupled to an NK cell effector agent.

“NK cell effector agents” are agents that cause proliferation,stimulation, adhesion to or activation of NK cells. NK cell effectoragents can be cytokines, adhesion molecules or NK cell activatingagents.

“NK cell activating agent” refers to stimulatory ligands that bind toactivating receptors present on the surface of NK cells.

“Modulate” or “modulating” as used herein refers to an increase ordecrease. Modulating results in any difference compared to normal immunefunction. Thus, modulating the immune system refers to increasing ordecreasing immune cells.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

As used herein, the term “subject” refers to any organism to which thedisclosed compositions can be administered, e.g., for experimental,diagnostic, and/or therapeutic purposes. Typical subjects includeanimals (e.g., mammals such as non-human primates, and humans; avians;domestic household or farm animals such as cats, dogs, sheep, goats,cattle, horses and pigs; laboratory animals such as mice, rats andguinea pigs; rabbits; fish; reptiles; zoo and wild animals). Typically,“subjects” are animals, including mammals such as humans and primates;and the like. Subjects can also refer to a cell or a cell line.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.In particular, in methods stated as comprising one or more steps oroperations it is specifically contemplated that each step comprises whatis listed (unless that step includes a limiting term such as “consistingof”), meaning that each step is not intended to exclude, for example,other additives, components, integers or steps that are not listed inthe step.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a membrane self-inserting peptide conjugate isdisclosed and discussed and a number of modifications that can be madeto a number of molecules including the membrane self-inserting peptideconjugate are discussed, each and every combination and permutation ofthe membrane self-inserting peptide conjugate and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Thus, if a class of molecules A, B, and C are disclosedas well as a class of molecules D, E, and F and an example of acombination molecule, A-D is disclosed, then even if each is notindividually recited, each is individually and collectivelycontemplated. Thus, is this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-group of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this application including, but not limitedto, steps in methods of making and using the disclosed compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

EXAMPLES A. Example 1: In Vive Generation of Cytotoxic Natural KillerCells and Associated Cancer Treatment Methods

The expansion of NK cells can occur by using membrane vesicles derivedfrom stimulator cells. K562-mb15-41BBL and K562-mb21-41BBL stimulatorcells were selected for generation of plasma membrane (PM) vesiclesbecause these cells were reported to expand NK cells very robustly anddo not require the isolation of NK cells from peripheral bloodmononuclear cell (PBMC) mixture prior to culture initiation.Furthermore, the presence of stimulatory ligands 41BBL and mbIL15 ormbIL21 can be easily tracked by antibody staining to confirm expressionof these molecules on the feeder cells and their presence in isolatedmembrane vesicles as well as on membrane coated microparticles. Whetheror not particles consisting of purified membrane vesicles derived fromstimulator cells support expansion of NK-cells in a similar fashion asthe stimulator cells do was tested. When PBMCs were exposed in cultureto 50 U/mL of IL-2 and increasing concentrations of PM-mb15-41BBL orPM-mb21-41BBL vesicles over 10 day period the NK cells content in thePBMC mixture increased proportionally to the PM concentration used(FIGS. 3 and 4 ). At the highest concentration of 200 μg/mL of plasmamembrane proteins tested, the resulting NK cell content was 70% and 82%in presence of PM-mb15-41BBL or PM-mb21-41BBL respectively. Forcomparison, the NK cells comprised only 22% of cellular content of thecultures that were exposed to IL-2 only. Furthermore, the total NK cellnumber increased in the cultures that were exposed to PM vesicles andthe increase was proportional to the PM concentration used. Thus, NKcell expansion was the highest in the cultures that received IL-2 with200 μg/mL of plasma membrane proteins where NK cells expanded 24 and 22fold over 14 days in the presence of PM-mb21-41BBL or PM-mb15-41BBLvesicles respectively while no expansion was observed in the controlculture (FIG. 5 ). Thus, both the level of expansion and content of NKcells in the culture were directly proportional to the concentration ofmembrane particles used (FIGS. 3, 4 and 5 ). Although the level ofexpansion in the presence of PM particles was lower than that observedin the control cultures where NK cells expanded by 88 and 150 fold inthe presence of live K562-mb15-41BBL and K562-mb21-41BBL, respectivelythis expansion is sufficient to generate the required doses for clinicalapplications. Thus, these experiments indicate that NK cells can beselectively expanded within PBMC mixture using particles consisting ofpurified plasma membrane vesicles with stimulatory ligands withoutfeeders.

NK cells expanded by culturing for 14 days with 200 μg/mL PM-mb15-41BBLor PM-mb21-41BBL membrane vesicles were tested for cytotoxic function ata 1:1 (E:T) ratio against chronic myeloid leukemia (CML) cell line K562,and acute myeloid leukemia (AML) cell lines KG1 and HL-60. Level ofcytotoxicity was compared to NK cells cultured with K562-mb15-41BBL orK562-mb21-41BBL live feeder cells (FIG. 6 ). NK cells stimulated withPM-mb15-41BBL and PM-mb21-41BBL killed 60 and 78% of K562 target cells,44% and 54% of KG1 cells and 9 and 15% of HL-60 respectively. Theselevels of cytotoxic response observed with PM stimulated NK cells wereonly slightly lower than those observed with NK cells cultured for 14days using the live K562-mb15-41BBL or K562-mb21-41BBL feeder celllines. These results indicate that NK-cells expanded using PM-mb15-41BBLare cytotoxic against acute myeloid leukemia targets and thus aresuitable for therapeutic applications in cancer treatment.

Together these results indicate that NK cells can be selectivelyexpanded within the PBMC mixture cells using membrane vesiclescontaining stimulatory ligands and in the absence of feeder cells. Thesecells are cytotoxic against various leukemia derived targets. Thismethod of expansion is amenable for both in vitro and in vivo expansionof NK cells for clinical applications. The suitability of membraneparticles for use in in vivo expansion is demonstrated by the increasedNK cell number in patients treated with dendritic cell derived exosomes(Dex) cancer vaccine-membrane particles naturally secreted by dendriticand other immune cells (Viaud, Terme et al. 2009).

FIG. 7 shows the generation of latex particles coated with PM-mb15-41BBLmembranes. FIG. 7 depicts histograms showing IL15-FITC (top) and41BBL-PE (bottom). Latex (left) or C18 Silica (right) beads were coatedwith K562mb15 plasma membrane containing membrane bound IL-15 and 41BBL.Both latex and C18 Silica bead showed presence of IL-15 and 41BBL aftercoating (Isotype in grey). To prepare the membrane-coated latexparticles, plasma membrane vesicles purified as described and containingthe stimulatory molecules 41BBL and mbIL15 were used and were embeddedon 5 micrometer latex microparticles using a previously establishedprotocol. Sulfate polystyrene latex microspheres (Inerfacial Dynamics)were resuspended in PBS, washed, and counted on a hemocytometer. To coatthe beads, an aliquot of bead suspension was added to PM-mb15-41BBLmembrane solution while sonicating the suspension. The bead-membranesolution was placed on a rotator at 4 degrees C. for 2 hours. Beads werecentrifuged and washed three times. The beads were labeled withantibodies and analyzed by flow cytometry to confirm the presence ofstimulatory proteins on the surface of the beads (left panels). Beadscoated with membrane showed positive staining for both IL-15 and 41BBLand low background staining with isotype control antibodies. C18-silicabeads were coated with PM-mb15-41BBL in a similar fashion. Again, thepresence of stimulatory molecules was confirmed by antibody staining(right panels). These results indicate that particles coated withcellular membranes containing stimulatory molecules can be generated.

FIG. 8 depicts debris found in cultures after NK cell expansion. a) FSCvs. SSC of PBMC (red) together with plasma membrane at day 10 ofculture. b) FSC vs. SSC of PBMC (red) together with K562mb15 feeder celldebris (green). The top and bottom figures represent separate cultureson day 10. The top figure has significantly more feeder cell debris vs.the bottom figure.

1. Materials and Methods

The cell lines K562-mb15-41BBL and K562-mb21-41BBL (K562-clone9.mbIL21)were used. K562, KG1 and HL-60 cell lines used in cytotox assays werepurchased from the American Tissue Culture Collection (ATCC).

Analysis were performed with the following mouse monoclonal antibodies:CD56-PE (Miltenyi Biotech), CD3-APC, CD16-FITC (Beckman Coulter),CD19-PECy7, 41BBL-PE (BD Biosciences) and 41BBL (R&D).

Other reagents and kits used include sulphate latex beads (5 μm;Invitrogen), C18-silica beads (10 μm; Sorbtec), RPMI media (ThermoScientific), CellGro media (Cellgenix) Fetal bovine serum (Invitrogen),IL-2 (Peprotec), Glutamax (Invitrogen). [0052] The preparation ofPM-mb15-41BBL and PM-mb21-41BBL plasma membrane vesicles was performedas follows. K562-mb15-41BBL and K562-mb21-41BBL were cultured in RPMImedia supplemented with 10⁰/FBS and the culture was scaled up to IL.Cells were harvested by centrifugation at 1000×g, washed with cold PBSwith 10 mM EDTA and resuspended in lysis buffer (50 mM HEPES, pH 7.4,protease inhibitor cocktail). Cells were disrupted with Douncehomogenizer and the solution was spun down at 300×g for 15 minutes toremove any remaining whole cells. The crude plasma membranes wereseparated from the cytosolic components by centrifugation for 30 min at4° C. (30000 rpm, 50Ti). The crude membranes were resuspended in lysisbuffer and further purified using a sucrose density gradient to yieldpure plasma membrane vesicles, referred to as PM-mb15-41BBL orPM-mb21-41BBL. A BCA assay was used to determine the membrane proteinconcentration while the amount of stimulatory 41-BBL ligand wasdetermined by Western blotting.

NK cell expansion within PBMC mixture was tested in the presence ofincreasing concentrations of PM-mb15-41BBL or PM-mb21-41BBL membraneparticles. The amounts used were 40, 80, and 200 μg of membrane proteinper 1 mL of culture which for PM-mb15-41BBL culture corresponded to 16,32, 80 ng of 41-BBL protein per mL of culture. PBMCs isolated from bloodby Ficol-Paque density gradient were grown in SCGM Cell Gro mediasupplemented with 10% FBS and 10 or 50 U/mL of IL-2 and increasingconcentrations of membrane particles. Cells were maintained at 37° C. ina humidified atmosphere with 5% CO₂. Starting on day 5 of culture mediawere exchanged every other day by replacing half of the media with freshmedia as well as replacing the amount of membrane removed through mediareplacement. Cells were counted every other day and the culture contentwas checked on days 7, 10 and 14.

Cytotoxicity assays were performed as follows. Leukemia derived targetcell lines K562, KG1, HR-60R were labeled with TFL4 (Oncoimmunin) orCellTrace Far Red (CTFR; Invitrogen), a far-red-fluorescent tracer forlong-term cell labeling, prior to addition of effector NK cells. Cellswere washed twice with PBS buffer with 1% BSA.

Target cells were then co-incubated with various amounts of NK-cells for90 minutes at 37° C. in the humidified atmosphere Air/CO₂ 95/5%. At theend of co-incubation cells were transferred to tubes, placed on ice andloaded with caspase/granzyme substrate (Pantoxilux kit, OncoImmunin) andlabeled with antibodies against surface antigens. Cells were immediatelyanalyzed by flow cytometry (BD FACS Canto-II). The NK cell-induced celldeath was detected using cell-permeable fluorogenic caspase/granzymesubstrate (OncoImmunin). Cleavage of this substrate by granzyme,released into the target cell upon interaction with NK-cells, anddownstream caspase produces green fluorescence inside dying targetcells.

B. Example 2: Optimization of Plasma Membrane Vesicles for Expanding NKCells 1. SUMMARY

Methods of preparing plasma membrane vesicles have been optimized aswell as methods of increasing the level of expansion of NK cells in thepresence of the vesicles. NK cells expanded with these methods weredetermined to kill primary leukemia cells from patients. The procedurewas scaled up for culturing K562-mb15-41BBL cells and optimizedpreparation of plasma membrane (PM) vesicles by disrupting cells usingnitrogen cavitation method. Optimized plasma membrane vesicles were morehomogenous and had higher levels of stimulatory ligand 41BBL. In the NKcell expansion tests with PBMC derived from healthy donors(n=3) asstarting material, the optimized PM vesicles outperformed previouspreparations of PM in stimulating NK cell growth and yielded levels ofselective NK cell expansion that are comparable to those attained withlive K562-mb15-41BBL feeder cells. The NK cell surface expression of amultitude of surface markers with relevance to NK cell function weredetermined in the staring material as well as in NK cells expanded inpresence of either PM vesicle or live feeder cells. The initial analysisindicated that the NK cells expanded with PM have similar activatedphenotype to those expanded with live K562-mb15-41BBL feeders cells yetsignificantly altered from the resting state phenotype as measured withcells freshly isolated from peripheral blood. The function of freshlyisolated and expanded NK cells were tested by measuring the cytotoxicitylevels against both leukemia cell lines (K562, KG1, HL-60, BDCM) andprimary leukemia blasts (from 3 different patient AML samples) atvarying effector to target ratios. The data indicate that NK cellsexpanded by stimulation with PM-mb15-41BBL vesicles kill cell lines andprimary AML cell and do it more efficiently than freshly isolated NKcells. In vivo testing in NSG of the NK cell expansion with PM has beenperformed and found that NK cells can be expanded directly in vivo inthe animal with serial dosing of PM vesicles.

2. RESULTS

NK expansion has been tested from four different healthy donors usingPM-mb15-41-BBL vesicle from the optimized plasma membrane preparations.The optimized PM-mb15-41-BBL vesicle efficiently and selectively expandsNK cells within the PBMCs mixture and the NK cell content increased withthe increasing amounts of PM vesicles (FIGS. 9, 10, and 11 ). The NKcell numbers increased on average 293 fold (range 104-557) after 12-13days of culture in the presence of the optimized PM vesicles and 173fold (79-895) in presence of live feeder cells (FIG. 12 ). Thisrepresents a significant improvement in the levels of NK cell expansionattained with the optimized PM vesicle over the initial preparations(233 vs. 23 respectively). The NK cells growth rate stimulated with PMvesicles changes over the 28 day culture time period with theexponential phase occurring between days 5 and 14 of culture after whichthe growth slows down (FIG. 13 ). Significant progress has been made inaccessing the cytotoxicity of the NK cells that were expanded using PMvesicle. NK cells expanded using optimized PM-mb15-41-BBL vesicleskilled all tested leukemia target cell lines as efficiently as NK cellsexpanded using live K562-mb5-41-BBL feeder cells (FIG. 14 ) and weresignificantly more cytotoxic as compared to freshly isolated NK cells(FIG. 15 ). Moreover, NK cells expanded with PM vesicle efficientlykilled patient derived CD34+ AML blasts while sparing healthy CD34−cells (FIG. 16 ). In humanized NSG mouse animal model, NK cellspreincubated for 7 days with PM-mb15-41-BBL vesicle and injected i.v.and further stimulated with PM-mb15-41-BBL vesicle delivered i.v. 3×weekly (M,W,F) along with low doses of IL-2 increased in numbers 15-20fold over 3 day period (FIG. 17 ) while control cells expanded underoptimal culture condition expended only 8 times over the same timeperiod.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

We claim:
 1. A method of treating cancer comprising administering to asubject an effective amount of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe NK cell effector agent is selected from the group consisting ofIL-15, IL-2, IL-12, IL-18, MICA, 2B4, LFA-1, or BCM1/SLAMF2, and whereinthe NK cell effector agent is not IL-21.
 2. The method of claim 1,wherein the composition comprises a membrane self-inserting peptideconjugated to IL-15.
 3. The method of claim 1, wherein the membraneself-inserting peptide is human Fc, GPI, transmembrane T cell receptor,or pHLIP.
 4. A method of expanding NK cells comprising administering toa cell population an effective amount of a composition comprising amembrane self-inserting peptide conjugated to an NK cell effector agent,wherein the cell population comprises NK cells and wherein the NK celleffector agent is selected from the group consisting of IL-15, IL-2,IL-12, IL-18, MICA, 2B4, LFA-1, or BCM1/SLAMF2, and wherein the NK celleffector agent is not IL-21.
 5. The method of claim 4, wherein thecomposition comprises a membrane self-inserting peptide conjugated to IL15.
 6. The method of claim 4, wherein the membrane self-insertingpeptide is human Fc, GPI, transmembrane T cell receptor, or pHLIP. 7.The method of claim 4, wherein administering to a cell populationcomprises administering the composition to a subject, wherein thesubject comprises the cell population.
 8. The method of claim 4, whereinthe composition further comprises a second membrane self-insertingpeptide conjugated to an NK cell effector agent, wherein the NK celleffector agent is selected from the group consisting of IL-15, IL-2,IL-12, IL-18, MICA, 2B4, LFA-1, or BCM1/SLAMF2.
 9. The method of claim8, wherein the two membrane self-inserting peptide conjugates areconjugated to the same or different NK cell effector agents.
 10. Amethod of modulating the immune system comprising administering to asubject one or more of a composition comprising a membraneself-inserting peptide conjugated to an NK cell effector agent, whereinthe NK cell effector agent is selected from the group consisting ofIL-15, IL-2, IL-12, IL-18, MICA, 2B4, LFA-1, or BCM1/SLAMF2, and whereinthe NK cell effector agent is not IL-21.
 11. The method of claim 10,wherein modulating the immune system comprises reducing the number ofactivated T cells.
 12. The method of claim 10, wherein modulating theimmune system comprises expanding the number of NK cells.
 13. The methodof claim 10, wherein the NK cell effector agent is IL-15.